植物和昆虫编码的miRNA已经被广泛证明存在甲基化修饰，并且甲基化修饰能够显著提高miRNA的稳定性和与AGO2蛋白的亲和性，从而影响miRNA的功能。本项目拟综合采用LC-MS/MS、RNA免疫共沉淀、氧化消除高通量测序、氧化消除定量PCR、氧化消除norther blotting等多种技术对哺乳动物细胞内miRNA的甲基化修饰，尤其是m6A甲基化修饰和3’末端2’-O-甲基化修饰进行系统性的鉴定，揭示METTL3负责miRNA m6A甲基化修饰和HENMT1负责miRNA 2’-O-甲基化修饰的分子机制，阐释miRNA m6A甲基化修饰和3’末端2’-O-甲基化修饰对miRNA的稳定性、与AGO2蛋白的亲和性和靶基因抑制效率的影响，探讨miRNA的这两种甲基化修饰可能的生理学和病理学意义。

The miRNAs encoded by plants and insects have been widely confirmed to be methylated, and the methylation can significantly improve the stability of miRNA and the affinity with AGO2 protein, thus affecting the function of miRNAs. This project intends to focus on the methylation of miRNAs in mammalian cells by LC-MS / MS, co-immunoprecipitation of RNA, high throughput sequencing of oxidative elimination, quantitative PCR of oxidation elimination, norther blotting of oxidation elimination and other technologies to systematically identify the m6A methylation and 3'-terminal 2'-O-methylation, and reveal the molecular mechanism of miRNA m6A methylation by METTL3 and 2'-O-methylation by HENMT1. This project will also try to elucidate the effect of miRNA m6A methylation and 3'-terminal 2'-O-methylation on the stability, affinity with AGO2 protein and efficiency of target gene suppression of miRNA, and investigate the possible physiological and pathological significance.